Alternative, more accurate methods exist

Bright-field microscopy is used to validate and quantify cell cultures. It gives ambiguous staining patterns and relies on a series of parameters, including roundness and uniformity, to discriminate cells from non-cellular particles.

Trypan blue stains dead cells in shades ranging from light blue to black, depending on the overall viability of the cell culture. When using a bright-field microscope, cells that are very lightly stained with trypan blue can be hard to differentiate from unstained cells, and thus hard to identify.

Also, individual lab members will each have a somewhat subjective view of what is a dead versus a living cell. Therefore, using trypan blue to distinguish between live and dead cells introduces the risk of variation between users and instruments.

For these reasons, fluorometric assays are considered a better choice for the evaluation of cell viability than colorimetric ones, such as trypan blue⁴.

Because cells have a relatively fixed amount of DNA/RNA, the DNA-interacting fluorophores acridine orange (AO) and 4′,6-diamidino-2-phenylindole (DAPI) will produce a uniform staining pattern for the quantification of cells, compared to the unspecified staining pattern when using bright-field based analysis.

AO is membrane-permeable and stains the total cell population, whereas DAPI only stains the dead cell population. Therefore, fluorometric determination of cell viability using AO and DAPI provides superior accuracy to identify live and dead cells with reduced noise and variation.

Under lock and key in EU laboratories

Recent changes have been made by the ECHA to highlight the fact that trypan blue is toxic to humans, and to restrict its use.

By classifying it as carcinogenic, the aim is to motivate users to phase out trypan blue use in laboratories across Europe⁵.

As of March 2021, this classification has already resulted in countries like Denmark requiring laboratories to have a permit to use trypan blue. The permit can only be granted if the use of it is strictly necessary in their workflow^6,7.

Furthermore, permit-holders are required to limit laboratory professionals’ exposure to trypan blue by ensuring a safe working environment through creating standard operating procedures (SOPs) for all workflows where trypan blue is used.

A part of this is to ensure that all samples and disposables exposed to trypan blue are collected and disposed of separately from normal laboratory waste.

Even though this currently does not apply to countries outside of Europe, it is generally agreed by the scientific community that there are risks associated with trypan blue5, implying that its use should be limited, as it may cause serious harm to users.

Alternatives to trypan blue

Alternatives to trypan blue include AO and DAPI which are not carcinogenic8,9. As alternatives to trypan blue, they can support a safer working environment.

Since the Via2-Cassette™ encapsulates these dyes, its design guarantees that any user contact with them is eliminated. This cassette technology can be readily established as a safety measure in laboratories across the world.

The Via2-Cassette™ is a unique microfluidic device that provides unparalleled accuracy and precision of a cell count and eliminates a user’s exposure to chemicals. The Via2-Cassette™ includes a built-in pipette that prevents errors during pipetting and staining.

The fluorescent dyes AO and DAPI are immobilized inside the cassette’s channels, automatically staining the total and dead cell populations, respectively.

During the entire cell counting process, AO and DAPI are kept inside the microfluidic device, significantly reducing the risk of exposure. The Via2-Cassette™ is disposed of after every use, helping to maintain a safe working environment.

The Via2-Cassette™ is used with the NucleoCounter® NC-202™, an automated fluorescent cell counter that provides an analysis of the total and live cells, the dead cell concentration, viability, and the DebrisIndex™ of a sample, all in approximately 30 seconds.

The instruments are constructed using high-quality components that remain stable over time, which together with extensive calibration procedures performed at the time of build, results in low variation between instruments, users and sites. This technology delivers an optimal level of precision to your cell counting.

The NucleoCounter® NC-202™ with the Via2-Cassette™, combines industry-leading accuracy and precision that delivers consistent results while keeping users safe from harm.

References

1. JR Tennant: Evaluation of the Trypan Blue Technique for Determination of Cell Viability. Transplantation, 1964. 2: p. 685-94.
2. KT Tsaousis, N Kopsachilis, IT Tsinopoulos et al.: Time-dependent morphological alterations and viability of cultured human trabecular cells after exposure to Trypan blue. Clin Exp Ophthalmol, 2013. 41(5):484-90.
3. AKH Kwok, C-K Yeung, TYY Lai, K-P Chan, CP Pang: Effects of trypan blue on cell viability and gene expression in human retinal pigment epithelial cells. Br J Ophthalmol. 2004;88(12):1590-4.
4. SA Altman, L Randers, G Rao: Comparison of trypan blue dye exclusion and fluorometric assays for mammalian cell viability determinations. Biotechnol Prog. 1993;9(6):671-4.
5. European Chemical Agency (ECHA): Substance Infocard: Tetrasodium 3,3′-[(3,3′-dimethyl[1,1′-biphenyl]-4,4′-diyl)bis(azo)]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonate]
6. Arbejdstilsynet: AT-vejledninger: Kræftrisikable stoffer og materialer
7. Retsinformation: Kræftbekendtgørelsen: Bekendtgørelse om foranstaltninger til forebyggelse af kræftrisikoen ved arbejde med stoffer og materialer
8. European Chemical Agency (ECHA): Substance Infocard: N,N,N’,N’-tetramethylacridin-3,6-yldiamine hydrochloride
9. European Chemical Agency (ECHA): Substance Infocard: 2-phenylindole-4′,6-dicarboxamidine dihydrohydrochloride (hydrate)